This proposal addresses the need for definitive evidence on the role of Foxp3+/T regulatory cells in the acceptance of physiologically relevant full MHC incompatible organ grafts in various settings of operational tolerance. In the initial year of funding (ARRA), spontaneous acceptance of MHC-mismatched mouse renal allografts was shown to depend exquisitely on Foxp3 cells using diphtheria toxin (DT) to transiently deplete circulating and intragraft Foxp3 cells in B6.Foxp3DTR mice bearing life sustaining DBA/2 renal allografts. The accepted grafts had distinctive nodular perivascular infiltrates of Foxp3 cells and dendritic cells (DC). Deletion of foxp3 cells caused dissolution of the nodular aggregates and widespread infiltration of the cortex, tubules and blood vessels by T cells, typical of acute rejection, and manifested by a rise in BUN over 7 days. Similar nodular aggregates of Foxp3 cells occur in grafts of patients who have accepted MHC mismatched kidneys on mixed chimerism tolerance induction protocols. Specific aim 1 will extend these observations to other forms of induced tolerance, mixed chimerism and neonatal tolerance, some of which are expected to be Foxp3 dependent; those with deletional mechanisms are expected to be Foxp3 independent. We will seek distinctive pathologic features in the graft, indicating Treg activity, such as the co-expression of latency associated protein (LAP) and Foxp3 or DC expression of indoleamine 2,3-dioxygenase (IDO), that might be used to distinguish foxp3 dependent from foxp3 independent forms of graft acceptance. The donor reactivity and regulatory potential of the cells in grafts and in lymphoid organs will be tested by ELISPOT and compared between the different forms of acceptance and acute rejection. The dependence of systemic tolerance on the intragraft infiltrates will be tested by graft nephrectomy in recipients with accepted kidney and skin grafts that have a remaining native kidney. Specific aim 2 will seek to identify the molecular and cellular basis for the dramatic strain differences in susceptibility to spontaneous graft acceptance, comparing BALB/c (rejected) and DBA/2 (accepted) kidneys (both H-2d) in B6 recipients. We will test for strain differences in DC function, cytokine/chemokine/complement mediators, minor histocompatibility antigens and ability to generate Foxp3 cells. Follow-up experiments seek to alter identified factors to promote acceptance of BALB/c kidneys. Specific aim 3 visualize interaction of Foxp3+ cells with other T cells, recipient and donor dendritic cells and tubular cells in accepting and rejecting renal grafts using a custom real time confocal, multiphoton endomicroscope. This system permits observations of the same graft over time, live video recording, delineation of 3-4 different cell types and computer assisted measurement of cell motility and interactions. These studies are designed to reveal the significance and in vivo function of Foxp3+ Treg in murine renal allograft acceptance and provide insights potentially applicable in clinical renal transplantation.